Melting temperature of molecular beacons as an indicator of the ligase detection reaction for multiplex detection of point mutations

Abstract

The multiplex detection of point mutations plays an increasingly important role in clinical diagnosis of genetic-based diseases. The present study reports a novel technology for the analysis of multiple point mutations based on the ligase detection reaction (LDR) and melting curve assay. In this method, the LDR was utilized to identify mutations using the high selectivity of Taq DNA ligase. While the probes match the target sequence perfectly, the allele-specific discriminating probe (5′ labeled with fluorophore) and common probe (3′ modified by quencher) will join with each other. Through the special design, the ligation product can then form a molecular beacon. Finally, the melting temperature (T_m) of the molecular beacon was measured by melting curve assay. The mutation type was identified through the melting curve and T_m value. For multiplex detection, molecular beacons with different T_ms were achieved by changing the length or GC content of the arm sequences and then presenting them to different mutation sites. Three common point mutations of the α-globin gene in thalassemia disease were identified simultaneously using this approach. The T_m achieved by the assay for each of the three mutation sites [Cd122 (C > G), Cd125 (T > C) and Cd142 (T > C)] was 73 °C, 71 °C and 60 °C, respectively. The veracity and reproducibility of the method was evaluated by clinical samples which were validated by sequencing. In summary, this method, based on the LDR and melting curve assay, provides a promising tool for detection of gene mutations.